We propose to exploit technologies and systems that we have developed to analyze in detail the molecular mechanisms involved with the somatic generation of the antibody repertoire. Specifically: 1. We have designed recombination substrate/gene transfer experiments to test details of the V gene assembly mechanism and similar types of experiments to elucidate the molecular mechanisms which regulate variable region gene assembly; a particular focus of the latter studies is the notion that tissue and stage specific assembly of VH, VL, and VT (T cell receptor) genes is regulated at the level of accessibility to a common recombinase. Ultimately, the mechanisms defined from the cell culture studies will be further elucidated by transgenic mouse methodologies. 2. We have demonstrated that germline VH gene expression occurs specifically in immature pre-B cells. The function of V gene expression will be investigated by elucidating the involved regulatory sequences, by searching for analogous expression of VL genes, and by identifying the nature and cellular location of putative VH proteins. 3. We have demonstrated that the most 3' VH gene segments are used highly preferentially by developing pre-B cells of Balb/c mice. To understand the role of this phenomenon in B cell development, we will extend these analyses to light chain V genes and to the development of the variable region gene repertoire in other mouse strains, including immunodeficient or autoimmune mice. We will also determine the factors which lead to normalization of the VH repertoire in adult mice. To facilitate these and other studies, we will continue to develop in situ RNA/RNA hybridization method to assay specific Ig gene expression in normal B lineage cells. To complete our analyses of VH gene usage, we will further define the structure and linkage of the most 3' VH family and the D families by a combination of cosmid cloning and pulsed field gradient gel electrophoresis methodologies. 4. Finally, we have identified a pre-B cell specific proto-oncogene; we will now use methods that we have developed to isolate, define, and characterize other gene products that are specific for the early stages of B cell development.